Evidence obtained from studies of a variety of neurotropic viruses and attenuated mutants identifies the major surface glycoproteins as determinants of viral neurovirulence. We have recently shown that mutants of murine hepatitis virus type-4 (MHV-4) contain internal sequence deletions and point mutations in the major spike glycoprotein, S, which render them neuroattenuated. Infections by these mutants are often restricted to limited subsets of CNS neurons and glial cells in vivo, and may result in chronic demyelinating disease characterized by episodes of demyelination, remyelination and further demyelination accompanied by mononuclear inflammatory responses. We propose to probe the molecular basis of neuroattenuation by characterizing the genetic changes in the S genes of deletion and point mutants of MHV-4. Additional neuroattenuated and neuroadapted mutants which we have isolated by selection for monoclonal antibody neutralization resistance and by passage in persistently infected murine olfactory bulb cells (OBL21A) respectively will be defined and the effects of these mutations on S function tested in vitro using vaccinia vectors expressing wild type and mutant S genes. Analysis of neural cell adapted virus derived by long-term culture in transformed mouse olfactory bulb neuronal cell cultures (OBL21A cells) has defined a heptad repeat region in the S2 posttranslational cleavage fragment of the S glycoprotein which is crucial for the expression by MHV of the property of pH independent cell fusion (polykaryon formation). Wild type MHV-4 fuses cells avidly at neutral pH and enter the cell by direct membrane fusion. Fusion negative mutants in contrast contain point mutations in this region and enter the cell exclusively via the acid endosome. In order to elucidate the effect of mutations in this heptad repeat region on spike stability and fusion, we will chemically synthesize model peptides based on the sequence of the heptad repeat region and analyze their structural interactions in vitro by gel exclusion chromatography, circular dichroism (CD) and two-dimensional nuclear magnetic resonance (2D-NMR). Finally, in immunologic studies the potential of CD4-and CD8-bearing T- lymphocytes to recognize and kill MHV-4 infected targets in vitro and to exert protective and virus clearing effects in vivo will be assessed by depletion and reconstitution techniques and by in vitro T-cell assays. Specifically addressed will be the potential of such CD4 and CD8 T-cells to restrict and clear persistent CNS infection and to contribute to the immunopathology of chronic demyelinating disease induced by MHV-4.